Tracing the path of disruption: 13C isotope applications in traumatic brain injury‐induced metabolic dysfunction

Abstract Cerebral metabolic dysfunction is a critical pathological hallmark observed in the aftermath of traumatic brain injury (TBI), as extensively documented in clinical investigations and experimental models. An in‐depth understanding of the bioenergetic disturbances that occur following TBI promises to reveal novel therapeutic targets, paving the way for the timely development of interventions to improve patient outcomes. The 13C isotope tracing technique represents a robust methodological advance, harnessing biochemical quantification to delineate the metabolic trajectories of isotopically labeled substrates. This nuanced approach enables real‐time mapping of metabolic fluxes, providing a window into the cellular energetic state and elucidating the perturbations in key metabolic circuits. By applying this sophisticated tool, researchers can dissect the complexities of bioenergetic networks within the central nervous system, offering insights into the metabolic derangements specific to TBI pathology. Embraced by both animal studies and clinical research, 13C isotope tracing has bolstered our understanding of TBI‐induced metabolic dysregulation. This review synthesizes current applications of isotope tracing and its transformative potential in evaluating and addressing the metabolic sequelae of TBI.

Cerebral metabolic dysfunction, a pathological phenomenon seen in experimental and clinical TBI studies, is a primary contributor to cognitive impairment. 4Immediately following primary injury, a transient period of cerebral hyperglycolysis occurs, 5 followed by a prolonged period of metabolic depression, which largely reflects impaired mitochondrial oxidative metabolism. 5Nevertheless, the observed perturbations in glucose utilization implicate alternative energy sources and bioenergetic reprogramming in the recovering brain post-trauma.Cerebral metabolic dysfunction induces rapid excitotoxicity wherein high extracellular glutamate concentrations disrupt homeostatic ionic gradients and membrane potentials within the parenchyma. 5,6This is followed by a cascade of events that includes impaired mitochondrial function, reactive oxygen species (ROS) formation, neuronal apoptosis, and necrotic cell death. 7The release of intracellular and intra-axonal contents from damaged cells can trigger inflammation by activating microglia 8 and recruiting peripheral immune cells. 9In addition, secondary injury caused by metabolic dysfunction also includes blood-brain barrier (BBB) breakdown and edema. 10search efforts to understand TBI-induced bioenergetic dysfunction may identify targets for novel interventions.A wide variety of experimental approaches have been used to measure metabolic pathways and bioenergetics. 13C isotope tracing is a powerful analytic approach that leverages precision quantification techniques like nuclear magnetic resonance (NMR), mass spectrometry (MS), and magnetic resonance spectroscopic imaging (MRSI) in tandem with molecular tracer technology to track the fate of labeled atoms from exogenous isotope-enriched substrate precursors through to their end metabolic products. 11is approach provides precise measurements of substrate flux through key pathways with high temporal resolution in a manner that surpasses alternative research methods, making it highly valuable for investigating metabolic changes in various organ systems and pathologies. 12,13Importantly, it has recently shown promise in studying TBI. 14 This review summarizes existing cerebral metabolic dysfunction research in TBI and introduces the applications of 13 C isotope tracing in this context.It further underscores this technique's relevance for future TBI research by emphasizing its prior uses in both experimental and human studies to identify areas of focus for future investigations.

| B I OENERG E TI C D IS TURBAN CE IN TB I
The brain is a highly aerobic and energy-demanding tissue, accounting for only 2% of total body mass while consuming around 20% of the body's daily energy expenditure. 15Early bioenergetic crisis is a common pathological occurrence in clinical and experimental TBI studies.The early post-TBI phase exhibits hyperglycolysis coupled with mitochondrial respiration depression. 16llular bioenergetics and mitochondrial functions are disrupted after the primary injury, which is particularly evident in the acute phase of moderate to severe TBI. 2,17Clinical evidence has established a clear link between cerebral bioenergetics dysfunction and outcomes, especially in moderate and severe TBI cases. 18perimental TBI models reveal a dynamic process of bioenergetic dysfunction, occurring within minutes or hours of injury and persisting for days, 19 closely tied to injury severity.The energetic crisis contributes to the activation of oxidative and neuroinflammatory stress pathways, 20 which are critical in driving the secondary brain damage cascades. 21,22Importantly, recent findings indicate that bioenergetic disturbances post-TBI can lead to iron homeostasis dysregulation and increase the susceptibility of cellular membranes to lipid peroxidation due to damaged mitochondria, impaired function of energy-dependent antioxidant systems, and increased ROS, thereby contributing to the onset and progression of ferroptosis. 23,24Thus, the energetic crisis following TBI emerges as a pivotal factor influencing outcomes, with bioenergetic dysfunction serving as a predictor for moderate or severe head injury prognosis. 25Understanding the pathological role of bioenergetic crisis in acute secondary brain injury after TBI holds significant importance.

| Mitochondrial dysfunction
Traumatic brain injury of all severities affects mitochondrial function. 21It has been reported that a substantial decline in mitochondrial respiration is observed as early as 30 min after severe TBI, 26 reaching peak mitochondrial oxidative damage and dysfunction at 12-24 h. 19,26Furthermore, mitochondrial complex 1-and complex 2-driven respiration shows a significant decrease at 1 and 3 h postmild, moderate, and severe injuries, with the impact proportional to injury severity. 27This underscores that mitochondrial impairment is a central aspect of metabolic dysfunction post-TBI and precedes various injury cascades, including oxidative stress. 28

| Glucose metabolic dysregulation
Glucose is a primary substrate for generating ATP under physiological conditions which is predominantly achieved through glycolysis and mitochondrial oxidative phosphorylation (OXPHOS). 29I triggers rapid metabolic dysregulation in the brain through unregulated ion release, mitochondrial damage, and molecular trafficking interruption. 30Studies in TBI animal models and headinjured patients reveal distinct triphasic patterns of cerebral metabolism: hyperglycolysis, metabolic depression, and metabolic recovery. 31Hyperglycolysis, marked by increased glucose uptake and lactate concentration, is an immediate response persisting for hours after TBI. 32,33Despite its association with enhanced energy production, hyperglycolysis correlates with worse neurological outcomes. 34This compensatory mechanism aims to restore the ionic gradient post-injury 17 and counterbalance mitochondrial dysfunction. 35Following this hyperglycolytic period, cerebral metabolism progresses to a prolonged period of metabolic depression, which features significantly reduced glucose metabolism and robust mitochondrial dysfunction, as documented in TBI experimental models 36,37 and in humans. 38,39The magnitude and duration of glucose metabolic depression is largely dependent on injury severity. 5The third and final phase is the metabolic recovery phase.It has been found that the recovery rate of the metabolic function is paralleled with that of neurobehavioral function. 31Notably, it has been found that complete recovery is commonly observed in mild TBI patients, but rarely seen after severe TBI. 40

| Lactate
After severe brain trauma, animal models show a significant rise in brain lactate levels, with immediate and continuous production following severe TBI. 41,42This increase is linked to multiple factors, including heightened astrocytic glycolysis due to tissue hypoxia, disrupted neuron-glia metabolic coupling, 43 activation of astroglia cytosolic malic enzyme, 41 and upregulation of lactate transporters in endothelial cells. 44Excessive lactate accumulation post-TBI correlates with poor prognosis in animal studies, 41,42 while low extracellular lactate levels are associated with better outcomes in clinical studies. 45Nonetheless, there is growing evidence that lactate metabolism aids cognitive recovery post-TBI, 46 acting as a mitochondrial respiration substrate. 47This reflects the brain's adaptive response to increased energy demands, highlighting lactate's therapeutic potential for TBI. 48,49However, the appropriate timing, dosage, and overall impact of lactate as an alternative energy source post-TBI are still debated. 50The relationship between lactate administration and TBI treatment may depend on the cerebral metabolism's triphasic metabolic pattern, suggesting that lactate administration during the hyperglycolysis phase could worsen outcomes, while its use during metabolic depression and recovery phases could be beneficial.This emphasizes the need for further research to pinpoint the optimal strategies for lactate use in TBI recovery.

| Pyruvate
Pyruvate, the product of glycolysis, can be converted into lactate or acetyl CoA.TBI has been found to inhibit both the expression and activity of pyruvate dehydrogenase (PDH) [51][52][53] in rats, potentially shifting pyruvate metabolism toward lactate.Clinical studies suggest impaired mitochondrial pyruvate metabolism in TBI, leading to decreased aerobic respiration at the location of injury. 54,55Elevated lactate-to-pyruvate ratio (LPR) in cerebral microdialysis is associated with unfavorable outcomes in TBI patients. 56Another human study with 223 TBI patients reported that pyruvate is a significant independent negative predictor of mortality. 45Pyruvate supplementation has been shown to improve TBI outcomes. 57,58

| Amino acids
Amino acids, essential building blocks of proteins, can function as alternative substrates for energy production, and their metabolism is reported to be dysregulated in TBI. 59Glutamate and glutamine are two important amino acids in the brain with relatively high concentration. 60The brain operates under a neuronal and glial metabolism coupling model, where glutamate released by neurons is taken up by astrocytes and converted into glutamine.Conversely, lactate released by astrocytes can shuttle to neurons. 61,62TBI leads to neuronal and glial metabolism uncoupling, 63 which can elicit excitotoxicity. 61,64Severe TBI patients also exhibit disturbances in cerebral aspartate metabolism. 65Derived from aspartate, N-acetyl aspartate (NAA) is a brain-specific metabolite and a sensitive marker of mitochondrial dysfunction and bioenergetics impairment. 66A body of evidence has shown that NAA is rapidly and substantially reduced in TBI patients 40,67 and models. 68,69Decreased NAA levels are proportionate to the degree of brain tissue damage post-TBI, attributed to reduced glucose metabolism and acetyl CoA, coupled with increased acetate metabolism in astrocytes. 67Additionally, compromised metabolism of branched-chain amino acids is linked to cognitive dysfunction after TBI, 70,71 with evidence suggesting their neuroprotective role. 72Dietary supplementation with branched-chain amino acids has been shown to ameliorate injury-induced cognitive impairment. 73

| Fatty acids
Fatty acids (FAs) serve as brain energy substrates, and dysregulated lipid metabolism significantly contributes to TBI pathophysiology and worsens neurological outcomes.Clinical studies indicate disturbed lipid metabolism predicts poor outcomes in severely injured trauma patients. 74Pre-clinical investigations suggest dysregulated brain lipid metabolism impairs cognitive performance after TBI. 50I leads to metabolism dysregulation of lipids and their downstream products, including FAs 75 and phospholipids. 76It has been reported TBI can increase oxidized free FAs, 75 a prominent product of lipid peroxidation and pro-inflammatory mediators. 75TBI also leads to dysregulation of FA metabolism.For example, TBI reduces l-carnitine, palmitic acid, and caprylic acid levels, while increasing acylcarnitine levels in the subacute phase. 77Medium-chain FAs, such as octanoic and decanoic acids, increase in severe TBI patients, 78 potentially influencing the energy crisis associated with mitochondrial failure in TBI. 79It is noteworthy that TBI increases FA oxidation to generate ATP in the subacute phase, 77 which reflects the brain's adaptive response to energy demands by shifting in fuel preference of FAs.In addition, TBI also leads to dysregulation of phospholipid metabolism, which is involved in the aggravation and expansion of neural tissue damage after TBI. 76,80

| Ketone bodies
Ketone bodies, including acetoacetate, β-hydroxybutyrate, and acetone, serve as natural alternative substrates to glucose for brain energy metabolism. 81Shifting toward ketone metabolism can limit the extent of cerebral injury, particularly when glucose utilization is compromised in TBI. 82,83Ketone bodies bypass the need for PDH to enter the tricarboxylic acid (TCA) cycle, like when PDH is inhibited in TBI. 52Administering exogenous ketones can help spare cytosolic NAD + pools, enhancing metabolic efficiency. 82Importantly, ketone metabolism is significantly higher in both the ipsilateral and contralateral sides of the injured brain after TBI. 84The energetically favorable mechanisms of ketone body metabolism under TBI conditions help alleviate cerebral energy deficits, 82 with increased cerebral uptake and oxidation of exogenous β-hydroxybutyrate improving ATP levels following TBI. 85

| Acetate
Acetate, an oxidizable substance in mitochondria, offers an alternative energy substrate for the brain, potentially aiding in the cellular response to TBI. 86 Disruptions in conventional energy metabolism in the injured brain may be mitigated by acetate, which is metabolized to acetyl-CoA by astrocytes.Studies suggest acetate administration may have neuroprotective effects in TBI, improving motor performance and cognitive outcomes and reducing neuronal damage in experimental models. 87While acetate metabolism is explored as a therapeutic target for TBI, ongoing research aims to understand its mechanisms, optimal dosages, and potential long-term effects in clinical settings.

| TR ADITIONAL ME TABOLOMIC TECHNI Q U E S
Various experimental approaches have enhanced our understanding of the metabolic pathophysiology of TBI in both models and human subjects (Table 1).Techniques such as arteriovenous gradient testing measure cerebral blood flow (CBF) and substrate consumption or release at specific time points following TBI. 25,88,89Cerebral microdialysis (CMD) allows in vivo collection of interstitial brain samples and enables focal medication administration. 34,45,902][93][94][95][96] MRSI can be used to measure and track the combined extra-and intracellular regional concentrations of endogenously labeled substances of interest (e.g.[106][107][108] A variety of biochemistry assays measure substances (e.g.0][111] Seahorse XF Analyzers can probe mitochondrial oxidative phosphorylation, glycolysis, and overall ATP production in live cells or tissues. 112Lastly, fluorescence microscopy with biosensors can measure specific compounds such as NAD + /NADH and cAMP. 113,114These technologies have been instrumental in establishing our understanding of cerebral metabolic disturbances in TBI.However, a major limitation of most traditional methods lay in that these technologies aren't capable of probing the granular details of complex pathways as they lack the resolution to study substrate turnover across time points. 115

| ISOTOPE TR ACING
While traditional metabolic measurement techniques focus on net substrate concentrations at specific time points, isotope tracing can infer metabolic flux over a defined period, measuring substrate flow through a pathway per unit of time. 116This technology, proven effective in various models and organ systems, 11,12,117,118 is crucial for characterizing the intricate chemical changes contributing to secondary brain injury (Figure 1, Tables 2 and 3).
Because flux itself isn't measurable, isotope tracing takes advantage of exogenously introduced fuel sources (Figure 2) that are labeled with naturally occurring stable isotopes of key organic atoms which don't radioactively decay (e.g. 1 H, 13 C, 15 N, or 31 P).As the body consumes these substrates through various metabolic pathways, the movement of the isotopic atom is trackable because it is incorporated into metabolites at specific locations in a pathway-dependent manner, each with a unique chemical signature that can be detected and quantified.Then, changes in the relative abundance of these metabolites over time can be used to infer overall flux through the said pathway.Strategic coupling of multiple tracers can infer complex metabolic activities, including glycolysis, TCA flux reversibility, gluconeogenesis, and other biosynthetic pathways. 11gnal detection and quantification in isotope tracing relies on various techniques, each with distinct advantages and limitations.
17][118][119][120] These established techniques provide detailed data on numerous substrates and metabolites in a sample.Recent computing advancements facilitate cross-referencing metabolite readouts to libraries, aiding in identifying unexpected pathway alterations. 121In clinical settings, sample procurement for these methods is often via CMD catheters or central venous lines, which carry notable risks. 56However, these access routes are commonly used in intensive care for routine monitoring, potentially imposing minimal additional risk.In cases where NMR or LC/GC-MS sample collection is restricted, recent studies highlight MRSI as a non-invasive and appealing alternative. 54,122SI additionally provides spatial context to the quantitative data that may be useful for better understanding the global metabolic TA B L E 1 Summary of traditional research methods for studying energy metabolism in TBI with example studies.changes that occur in the brain following trauma, which the former detection methods typically lack.Therefore, the detection method should be carefully selected considering safety concerns, logistical constraints, and the overall objectives of the individual study employing isotope tracing.

Methods
4.1 | 13 C-glucose tracing 13 C-labeled glucose tracers are widely employed in models 33,41,86,[123][124][125][126][127][128] and human studies 106,129 of metabolic dysfunction in TBI (Figure 2).One notable example is 1-13 C-glucose, which allows accurate tracing of its single labeled carbon molecule through glycolysis and the TCA cycle (Figure 3).Due to innate differences in metabolic biochemistry in neurons versus astrocytes at several steps, this tracer is additionally able to partially discern contributions to overall cerebral glucose metabolism made by the astrocytic compartment.As such, it has proven useful for evaluating the interplay between anaerobic respiration and oxidative metabolism in the brain following trauma. 47,86,123However, because the C1 carbon of the glucose backbone is lost as CO 2 in the hexose to pentose conversion step of the pentose phosphate pathway (PPP), which serves as a vital source of antioxidants for combating the increased ROS production in brain injury, 130 this tracer is poorly suited to track PPP metabolic flux, a major limitation of its utility in TBI.
To circumvent this, glucose tracers with an additional labeled carbon, such as 1,2-and 1,6-13 C 2 -glucose, may be employed.26][127][128][129]131 The addition of a labeled carbon in the C2 or C6 position allows for the detection of 1-or 5-13 C-ribulose-5-phosphate signals when glucose enters the oxidative PPP, respectively.The movement of these metabolites can then be followed back into glycolysis via the production of monolabeled fructose-6-phosphate (e.g.1-13 C-fructose-6-phosphate) and subsequent downstream metabolites which aren't produced by glycolytic metabolism of dilabeled glucose. 11,132Alternatively, the absence of this signature concurrent with reduced monolabeled ribulose-5-phosphate levels 13 C isotope tracing of metabolism in TBI. 13 C tracer can be administered in vivo to animals or human subjects, ex vivo to fresh tissue, or in vitro to culture model systems (1).Metabolites are extracted from the target tissues or fluids or cells at specified intervals post-injury (2) and detected using analytic techniques including NMR, mass spectrometry, and MRSI (3).Finally, analyte readouts are analyzed and referenced to metabolite libraries to quantify pathway flux (4).MRSI, magnetic resonance spectroscopic imaging; NMR, nuclear magnetic resonance.
TA B L E 2 Summary of studies using 13 C isotope tracing to assess metabolic response to TBI in model systems.indicates the pentose products of the PPP are instead being used for nucleotide synthesis. 114][125][126][127][128][129] This technique also robustly supports the acute diversion of glucose into the PPP, supporting the theory inflammatory cascades induce early hyperglycolysis, causing significant ROSmediated damage and necessitating increased NADPH production for protection. 106,125,127,129,131Additionally, despite the initial hyperenergetic phase, a significant reduction in overall anaplerotic flux at 24 h, coupled with an increased pyruvate carboxylase/PDH ratio, implies a shift in glucose usage from energy production to regenerative processes later in secondary injury. 124Hence, glucose tracers play a crucial role in confirming and extending existing knowledge while identifying new directions for glucose-mediated cerebral metabolomics research in TBI.

| 13 C-pyruvate tracing
Labeled pyruvate enables the differentiation between oxidative and anaerobic consumption of glycolytic metabolites.It directly assesses pivotal enzyme activity, such as PDH, linking glycolysis to mitochondrial OXPHOS, and lactate dehydrogenase (LDH), a source of ATP production independent of mitochondrial respiration. 133portantly, the cerebral extracellular LPR, a well-studied measure in neurocritical care, consistently correlates with increased mortality and morbidity in TBI patients. 134,135However, while valuable for prognosis, LPR alone doesn't capture the molecular conditions underlying altered lactate production or consumption, as discussed later.
The utilization of 1-13 C-pyruvate has shed light on post-TBI mechanisms 54,55,122,136,137 (Figure 2), revealing how 13 C-bicarbonate production, an OXPHOS activity marker, 136 aligns with 13 C-pyruvate mitochondrial consumption.Impaired mitochondrial function is marked by reduced bicarbonate and increased 13 C-lactate levels due to a shift toward anaerobic respiration via LDH.Studies in both animals and humans post-TBI have consistently found significant mitochondrial dysfunction from the acute phase to at least a week post-injury, necessitating a switch from oxidative to anaerobic respiration to meet cerebral energy needs. 54,55,122,136,137One study using hyperpolarized 1-13 C-pyruvate demonstrated these changes are detectable in repetitive TBI brains extending beyond 3 months post-injury. 137Others have utilized it to explore aspects of pyruvate metabolism in the injured brain in closer detail.For example, depleting microglia before TBI induction lessens pyruvate metabolism dysregulation, highlighting the significant role of microglial activation in these bioenergetic changes. 122Furthermore, dichloroacetate coadministration, a PDH kinase inhibitor, showed no difference in PDH activity, indicating aerobic metabolism reduction is not due to PDH kinase inhibition. 53Interestingly, all studies using 1-13 C-pyruvate to date have employed MRSI as the detection method, highlighting its potential as an early, noninvasive imaging modality for identifying cerebral metabolic changes in otherwise radiographically normal TBI patients. 55rther, the use of such technology in tandem with machine learning workflows was recently shown to be capable of predicting long-term behavior changes after TBI in animals that otherwise lack detectable changes in conventional imaging at the same timepoint, 137 which suggests isotope tracing may be a powerful approach to better study the link between TBI and the elevated risk of developing long-term neurobehavioral sequelae including mental health disorders 138 and dementia, 139 among others.However, it is critical to note that the application of hyperpolarized 1-13 C-pyruvate in TBI models has shown a rapid lactate increase at the injury site, 136 potentially worsening the focal injury and posing limitations for its use in TBI patient studies, and this must be carefully considered when designing experiments or clinical procedures.

| 13 C-lactate tracing
Lactate serves as a viable neuron fuel and participates in intricate homeostatic interactions between excitatory neurons and astrocytes. 140Recently, clinical studies using labeled lactate have sought to better understand neurons and astrocytes activity following trauma and its relation to patient prognosis. 62,141,142A study using 3-13 C-lactate tracing was the first to demonstrate the brain's capability of using lactate as a fuel source following head trauma 62 and it was later shown that lactate is utilized differently in the post-traumatic brain compared to controls in humans 141 (Figure 2).This indicates situational lactate metabolism can compensate for the reduced glucose processing capability of the brain in the depressed metabolic phase of secondary injury.Further, additional studies have demonstrated peripheral lactate mobilization is at least partly responsible for the systemic hyperglycemia that is known to occur following trauma 143 and that lactate-derived carbohydrate substrates either directly, via oxidative metabolism of lactate, or indirectly, via peripheral gluconeogenesis, provide the majority of metabolic energy to the brain after TBI. 142vertheless, severe TBI can render neurons incapable of using F I G U R E 3 Labeling patterns for energetic metabolism of the tracer 1-13 C-glucose.Labeled glucose may either be converted to pyruvate via glycolysis or shunted into the PPP.The produced labeled pyruvate may either be metabolized anaerobically via LDH or enter the TCA cycle via PDH (neuronal compartment) or PC (astrocytic compartment).OAA, oxaloacetate; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; PPP, pentose phosphate pathway; TCA, tricarboxylic acid; αKG, α-ketoglutarate.
lactate as an alternate fuel source to meet metabolic needs, which can focally elevate lactate concentrations to a degree that may damage or even kill local tissues. 41Hence, lactate's role in the brain in health and injury is complex, and future lactate tracing studies can directly probe its function as both a waste product and fuel source.

| 13 Cβ -hydroxybutyrate and succinate tracing
Several additional tracers have been used in the setting of TBI. 84,144,145As PDH may be directly inhibited by ROS, PDH dysfunction is one of the commonest causes of mitochondrial dysfunction and subsequent OXPHOS impairment in trauma. 146The ketone body β-hydroxybutyrate (β-HB) can be directly converted to acetyl-CoA, independent of PDH activity, making labeled β-HB suitable for assessing mitochondrial enzyme activities within the TCA cycle. 84In a TBI model, 2,4-13 C 2β-HB tracer revealed increased ketone metabolism enhancing TCA and subsequent OXPHOS activity in both neurons and astrocytes despite PDH dysfunction 84 (Figure 2).Succinate metabolism via succinate dehydrogenase provides electrons to the electron transport chain (ETC) independently of NADH reduction via mitochondrial complex I. 147 Focal 2,3-13 C 2 -succinate administration improved cerebral TCA functioning by bypassing complex I, supporting ETC activity. 144,145While the impact on patient outcomes remains uncertain, these studies offer unique insights into oxidative glucose metabolism dysfunction post-injury and warrants additional studies.

| Combination tracing
Combination of multiple labeled tracers can enhance understanding of cerebral bioenergetic dysregulation in TBI, elucidating contributions from distinct cell populations and specific metabolic aspects.Co-administering 1-13 C-glucose and 1,2-13 C 2 -acetate in experimental TBI revealed greater reductions in oxidative glucose metabolism in neurons than astrocytes (Figure 2).Astrocytes, in response to TBI, utilize acetate to elevate glutamate concentrations through the glutamate-glutamine cycle, attempting to restore metabolic function post-injury. 86By comparing TCA metabolite labeling patterns from animals given either 1-13 C-glucose, 3-13 C-lactate, or 2-13 C-acetate, it was further shown neuronal metabolic dysfunction becomes entirely uncoupled from the metabolic activity of the astrocytic compartment within minutes of severe TBI, demonstrating that astrocytic lactate production continues despite impaired neuronal consumption of the substrate. 41ese examples highlight only a few examples of how tracer combinations can unravel complex metabolic changes and compartmental contributions in secondary injury.or by isolating brain cells post-TBI following 13 C isotope infusion.Yet, relying solely on 13 C isotopes may not completely reveal the metabolic processes behind the secondary injuries of TBI.Other tracers, each with unique benefits for dissecting TBI pathology, complement the capabilities of 13 C isotope tracer.For instance, 18 F-fluorodeoxyglucose offers insights into glucose metabolism, 149 while 11 C-Pittsburgh compound B targets amyloid deposits, illuminating post-TBI neurodegenerative processes. 150Integrating 13 C with other isotope tracers like 1 H, 15 N, or 31 P, commonly utilized in TBI studies across humans and animals, could enable a more thorough investigation of cerebral metabolic disturbances. 60Furthermore, the development and application of novel 13 C tracers remain crucial for advancing our understanding of post-trauma cerebral metabolic dysregulation.

| CON CLUS I ON S AND PER S PEC TIVE
For instance, using 13 C octanoate, a BBB-permeable mediumchain FA constituting a significant portion of the normal free FA pool, could offer insights into brain FA metabolism. 151Employing 13 C isotope tracing for amino acids such as glutamine, aspartate, and branched-chain amino acids will deepen our comprehension of amino acid metabolism in TBI. 12 Exploring new therapeutic avenues for TBI through 13  In summary, the application of 13

CO N FLI C T O F I NTE R E S T S TATE M E NT
Dr. Xiaoying Wang is an Editorial Board member of CNS Neuroscience and Therapeutics and a co-author of this article.To minimize bias, he was excluded from all editorial decision-making related to the acceptance of this article for publication.The authors declared no potential conflicts of interest with respect to the authorship of this article.
C isotope tracing, by elucidating specific metabolic pathways and dysfunctions, could identify novel therapeutic targets.Pinpointing areas of mitochondrial dysfunction or abnormal glucose, amino acids, and fatty acids metabolism may lead to interventions designed to restore metabolic balance and mitigate secondary injury. 152Additionally, 13 C tracing technologies may facilitate personalized treatment strategies, enabling clinicians to customize interventions based on individual metabolic profiles, potentially enhancing outcomes.The application of isotope tracing may also provide a method to evaluate the efficacy of therapeutic interventions in real time, with changesin tracer uptake or metabolite levels signaling whether a treatment is effectively addressing metabolic dysfunction or improving cerebral blood flow post-TBI.153,154 Figures were prepared using the tool suite available at BioRe nder.com (https:// app.biore nder.com/ biore nder-templ ates).

Principle Utility in TBI Animal studies Clinical studies
Summary of studies using 13 C isotope tracing to assess metabolic response to TBI in human subjects.
13hematic of fuel utilization and13C-isotope tracing in TBI studies.